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The effect of a linseed oil diet on hibernation in yellow-bellied marmots (Marmota flaviventris)
Physiology and Behavior 68 (2000) 431–437
Original articles
The effect of a linseed oil diet on hibernation in yellow-bellied marmots (Marmota flaviventris) Vanessa L. Hill, Gregory L. Florant* Colorado State University, Department of Biology, Ft. Collins, CO 80523, USA Received 22 December 1998; received in revised form 5 August 1999; accepted 26 August 1999
Abstract The essential fatty acids (EFAs), ␣-linolenic acid (18:3,n-3) and linoleic acid (18:2,n-6) are known to be important for mammalian hibernation. In marmots (Marmota flaviventris), reducing both dietary EFAs alters hibernation patterns by causing an increase in energy expenditure, but hibernation still occurs. In this study, marmots fed a diet high in ␣-linolenic acid, with normal linoleic acid levels, had significantly (p ⬍ 0.05) more ␣-18:3 in their WAT and plasma unesterified fatty acids after 4 months than did marmots fed a control diet. During the winter, the control marmots hibernated normally while the marmots fed the ␣-18:3 diet did not hibernate, continued to eat, and lost less mass than the control group during the winter. These results suggest that ␣-18:3 may play a role in regulating normal hibernation behavior in marmots. © 2000 Elsevier Science Inc. All rights reserved. Keywords: Hibernation; Essential fatty acids; ␣-Linolenic acid; White adipose tissue; Plasma
1. Introduction The polyunsaturated, essential fatty acid, linoleic acid (cis-9,12 octadecadienoic acid or 18:2,n-6) is important for normal hibernation in mammals that hibernate (i.e., hibernators). Linoleic acid is considered essential because mammals cannot synthesize it, and linoleic acid is essential for normal reproduction, skin maintenance, and other physiologic functions [1,2]. Hibernators that do not obtain sufficient 18:2 in their diet during the summer feeding period store less 18:2 in their white adipose tissue (WAT) and arouse more frequently, have shorter torpor bouts and higher metabolic rates during hibernation than hibernators with sufficient dietary 18:2 [3–8]. These changes in hibernation behavior increase energy expenditure, which might increase the rate at which endogenous energy reserves (i.e., WAT) are depleted, and jeopardize the hibernator’s over winter survival. The WAT of free-ranging hibernators contains another, essential, polyunsaturated fatty acid, ␣-linolenic acid (cis-9,12,15 octadecatrienoic acid or ␣-18:3,n-3) [9–11]. In previous hibernation studies, the diets contained very little ␣-18:3 (⬍3% of total lipids). Because hibernators cannot synthesize ␣-18: 3, the effects of ␣-18:3 in the diet and WAT of hibernators has not been investigated in laboratory experiments [3, 4, 6– * Corresponding author. Tel.: 970-491-7627; Fax: 970-491-0649 E-mail address: [email protected]
8]. Consequently, very little is known about how ␣-18:3, and the longer chain, polyunsaturated fatty acids (n-3, PUFAs) synthesized from ␣-18:3, are stored and utilized for hibernation. Furthermore, it is not known if changes in dietary ␣-18:3, like 18:2, will result in changes in behavior (i.e., torpor bout patterns) during hibernation. In this study, we fed yellow-bellied marmots (Marmota flaviventris) a linseed oil diet with a concentration of ␣-18:3 similar to the leafy portions of plants that are the main components of their natural diet [9]. We compared this group to a group of marmots fed a control diet. Marmots are particularly good animal models to study the effect of dietary fatty acids on hibernation because they do not eat during the winter and rely entirely on fat stored from summer feeding [3, 12]. The first objective of this study is to determine the seasonal patterns of fatty acid composition of WAT and plasma unesterified fatty acids in hibernators fed a diet rich in ␣-18: 3. The WAT composition represents the storage of fatty acids and the plasma unesterified fatty acids are a measure of fatty acid utilization. We hypothesize that ␣-18:3 will be stored in WAT during the summer and preferentially retained in WAT during hibernation as has been shown to happen with the other essential PUFA, 18:2 [3,9]. The second objective of this study is to determine what effect a diet rich in ␣-18:3 has on hibernation behavior as measured by torpor bout length and body temperatures (Tbs). Because we know that the essential PUFA 18:2 in the diet of hibernators results in longer torpor bouts and lower Tbs during hiberna-
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tion, we expect that ␣-18:3, which is also an essential PUFA, will have a similar effect. Specifically, we hypothesize that marmots fed the linseed oil diet will have longer torpor bouts and/or lower Tbs than marmots fed a control diet.
2. Materials and methods 2.1. Animals Nine adult marmots of both genders were trapped in Colorado during May and June of 1996, and immediately brought into the lab. These marmots were randomly placed in either the control or linseed oil diet group. From a group of four marmots that had been in the lab for 1 year, two were randomly placed in each group. The control diet group consisted of five marmots fed Purina 5001 and the other group consisted of eight marmots fed a 4.5% linseed oil (Purina 5806-I). Body mass was measured monthly throughout the study. The linseed oil diet was designed to have levels of ␣-18: 3 acid that were higher than the control diet, but comparable to the marmots’ natural diet. The diets were nutritionally equivalent except for the composition of the fat (see Table 1). Both diets were made of all natural ingredients, and meet the requirements established by Purina Research and the National Academy of Sciences (Purina, personal communication). The form of linseed oil used in our diet is standard for many experimental diets, and is safe and nontoxic. The antioxidant, ethoxyquin, was added to the diet to prevent oxidation of fatty acids. The linseed oil diet was also kept frozen or under refrigeration until fed to the marmots to further retard the oxidation of fatty acids. 2.2. Summer conditions Animals were caged individually at 25⬚C under a longday light cycle (14L:10D) during the summer (April–September). Both food and water were provided ad lib. Food intake was measured on a weekly basis. Each day the amount of food fed to each marmot was weighed. Twice per week the food left in the cage (including spillage) was weighed. The amount of food fed each marmot was summed for the week and the amount left in the cage for that week was subtracted. This total was then divided by 7 to calculate the average daily intake.
2.3. Winter conditions In October, the marmots were moved to a 5 ⫾ 3⬚C cold room and complete darkness. Cages for control and linseed oil diet marmots alternated in the cold room. Average daily food intake was calculated every 2 weeks to minimize disturbance to hibernating animals. Marmots were fed every 3 to 4 days, and leftover food was removed once every 2 weeks. 2.4. White adipose tissue and plasma sampling The WAT of each marmot was sampled three times: at capture (Apr–June), prior to hibernation (October), and immediately after hibernation (March). Tissue samples were taken by making a small 1⬙ incision off midline into the abdominal cavity after the marmot had been anesthetized with ketamine (32 mg/kg) mixed with acepromazine (10 mg/kg). A small sample of WAT (⬍1 g) from either the gonadal or omental fat pad was biopsied and immediately placed under nitrogen and stored in a freezer at ⫺80⬚C. The triacylglycerol content and composition of gonadal and omental fat deposits are very similar [9]. Therefore, samples from either depot were used. A blood sample was taken from the femoral artery or vein at the same time that WAT was sampled. The blood was centrifuged, and the recovered plasma stored in a nonheparinized tube in a freezer at ⫺80⬚C. Marmots were allowed 1 week to recover after surgery at 25⬚C (14L: 10D). All procedures were approved by the Colorado State University Animal Care and Use Committee. 2.5. Body temperature/torpor bout measurements In October, temperature sensitive transmitters encased in paraffin (Minimitter, Model T-M Disc with a 2000MAH lithium battery) were surgically implanted into the abdominal cavity of each marmot when WAT and plasma samples were taken. For each animal, the transmitter emitted a specific frequency that was carried via an individual antenna to a computer outside the cold room where the frequencies were converted to body temperatures (Tb) and stored. Body temperatures were recorded for each animal every 30 min from November to March 1996. 2.6. Analysis of WAT and plasma samples Fatty acids were extracted and methylated from a 10–20mg sample of WAT [13]. Separation of fatty acids was
Table 1 Comparison of fatty acid composition of the control and linseed oil experimental diets and leaves from Taraxacum officinale, which is a main component of the marmots’ natural diet Fatty acid (% of total) Diet Control Linseed oil T. officinale
14:0
16:0
16:1
18:0
18:1
18:2
␣-18:3
Other
% Fat
2.1 0.3 0.4
21.8 7.6 14.8
3.8 0.04 2.3
9.5 3.7 1.4
33.4 19.7 2.2
26.1 17.5 17.9
2.1 50.9 58.9
1.2 0.2 1.9
4.5 4.5 3.6
Means are the results of three trials per diet. Percent of each dietary component for control/linseed oil diet: protein 23.4/23.5, fat 4.5/4.5, fiber 5.3/5.3, carbohydrate 66.5/66.4; kcal/g 3.3/3.7 (Purina Test Diets, Inc.).
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achieved with a packed column (10% SP2330 100/120 chromosorb WAW, Supelco, Inc.) on a Hewlett-Packard 5890 gas chromatograph. The temperature of the column was 100⬚C at injection, held there for 1 min, and then the temperature was increased by 5⬚C every minute until it reached 260⬚C, after which time all fatty acids had been eluted. All fatty acids were identified using standards from Matreya, Inc. (Pleasant Gap, PA). Plasma unesterified fatty acids were extracted and methylated as previously described [9], with one modification: unesterified fatty acids were separated from other plasma lipids using a solvent mixture of 80 hexane:20 ethyl ether:2 formic acid [14]. The unesterified fatty acid methyl esters were identified by GC in the same manner as the WAT fatty acids. Unesterified fatty acid concentrations were determined by 50 nmol pentadecanoic acid (15:0) added as an internal standard to each sample.
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ids (PUFA, n-6) — 18:2, 20:3, 20:4, 22:4; polyunsaturated n-3 fatty acids (PUFA n-3) — ␣-18:3, 20:5, 22:5, 22:6. A two-way ANOVA, with diet and time as independent variables, was applied to each class of fatty acids (i.e., SFAs, MUFAs, etc.) in WAT and plasma unesterified fatty acids. If the overall ANOVA model was found to be significant for a particular class of fatty acids, significant differences between time and diets were detected using pairwise comparisons of least squares means. Five preplanned comparisons were used: capture (control diet versus linseed oil diet), capture versus prehibernation (control and linseed oil diet), and prehibernation versus posthibernation (control and linseed oil diet). The significance level of these comparisons was modified with a Bonferroni adjustment (p ⫽ 0.05/5 ⫽ 0.01).
2.7. Identification of fatty acids The following fatty acids were identified in varying amounts in WAT, plasma unesterified fatty acids and the diets: the saturated fatty acids (SFAs) — tetradecanoic acid (14:0), hexadecanoic acid (16:0), octadecenoic acid (18:0), and eicosanoic acid (20:0); the monounsaturated fatty acids (MUFAs) — 9-hexadecanoic acid (n-7,16:1) and 9-octadecenoic acid (n-9,18:1); the polyunsaturated n-6 fatty acids (n-6 PUFAs) — 9,12-octadecadienoic acid (18:2), 8,11,14eicosatrienoic acid (20:3), 5,8,11,14,17-eicosatetraenoic acid (20:4), and 7,10,13,16-docosatetraenoic acid (22:4); the polyunsaturated n-3 fatty acids (n-3 PUFAs) — 9,12,15octadecatrienoic acid (␣-18:3), 5,8,11,14,17-eicosapentaeoic acid (20:5), 7,10,13,16,19-docosapentaenoic acid (22: 5), and 4,7,10,13,16,19-docosahexanoic acid (22:6). 2.8. Hormone analysis Testosterone levels and estradiol levels were analyzed by Dr. Gordon Niswender and the Endocrine Laboratory of Animal Reproduction and Biotechnology [15,16]. 2.9. Statistics All statistical analyses were done with the SAS program (SAS Institute). All data were checked to be sure that the assumptions of the models were not violated. Food intake and mass data between the two diet groups were compared using a repeated measures analysis of variance design. The first 2 months of body mass data were not used in the analysis because the sample sizes for each group were changing as newly trapped animals were added. For this reason, and the fact that the animals needed an additional acclimation period to the lab, the first 3 months of food intake data were not used. For statistical analysis, fatty acids from WAT and plasma were combined into four categories: saturated fatty acids (SFAs) — 14:0,16:0,18:0,20:0; monounsaturated fatty acids (MUFAs) — 16:1, 18:1; polyunsaturated n-6 fatty ac-
Fig. 1. Fatty acid composition of the WAT of control (A) and linseed oil (B) fed marmots at capture (April–June), prior to hibernation (Oct.) and after hibernation (March). All points represent the mean ⫾ 1 SE. Saturated fatty acids (SFAs) included primarily 16:0 with ⬍5% of 14:0 and 18:0 combined. Monounsaturated fatty acids (MUFAs) were largely 18:1(n-9), with ⬍5% 16:1 (n-7). n-6 Polyunsaturated fatty acids (n-6, PUFAs) were entirely 18:2, and n-3 PUFAs were entirely ␣-18:3 (n-3). *Indicates a significant difference from the previous time period. Control marmots n ⫽ 5 and linseed oil diet marmots n ⫽ 8.
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3. Results The WAT fatty acid composition of the control diet group (Fig. 1a) did not differ significantly from the linseed oil diet group (Fig. 1b) at capture. Between capture and prehibernation, several changes in the fatty acid composition of WAT occurred in both diet groups. The saturate percentage of total WAT fatty acids decreased significantly in the linseed oil diet group [ANOVA, diet ⫻ time, F(2,33) ⫽ 13.11, p ⫽ 0.0001; capture versus prehibernation p ⫽ 0.0001], but did not change in the control group. The monounsaturate percentage of total WAT fatty acids increased significantly in the linseed oil diet group [ANOVA, time, F(2,33) ⫽ 14.17, p ⫽ 0.0001; capture versus prehibernation p ⫽ 0.0003, but did not change in the control group. Linoleic acid (18:2,n-6) was ⬎99% of all polyunsaturated n-6 fatty acids (n-6, PUFA) in the WAT of both diet groups. The percentage of total WAT fatty acids comprised of 18:2 decreased significantly in the linseed oil diet group [ANOVA, diet ⫻ time, F(2,33) ⫽ 12.42, p ⫽ 0.0001; capture versus prehibernation p ⫽ 0.0001]), but remained the same in the
Fig. 2. Changes in classes of unesterified fatty acids in plasma of control (a) and linseed oil diet (b) marmots. All points represent the mean ⫾ 1 SE. *Indicates a significant difference from the previous time period. Saturated fatty acids (SFAs) included 14:0, 16:0, 18:0, and 20:0. Monounsaturates (MUFAs) were the same as described in Fig. 4. n-6 Polyunsaturated fatty acids (n-6 PUFAs) were primarily 18:2, with ⬍50 mol/L of 20:3, 20:4, and 22:4 combined. n-3 PUFA fatty acids were primarily ␣-18:3 with ⬍10 mol/L of 20:5, 22:5, and 22:6 combined. Sample sizes are the same as listed in Fig. 4.
control group. Alpha-linolenic acid (␣-18:3,n-3) was ⬎99% of all polyunsaturated n-3 fatty acids (n-3, PUFA) in the WAT of both diet groups. The percentage of total WAT fatty acids comprised of ␣-18:3 decreased significantly in the control group [ANOVA, diet, F(2,33) ⫽ 12.82, p ⫽ 0.001, time, F(2,33) ⫽ 12.88, p ⫽ 0.0001; capture versus prehibernation p ⫽ 0.005], but did not change significantly in the linseed oil diet group. No significant changes in the fatty acid composition of WAT occurred between prehibernation (Oct.) and posthibernation (March) in either diet group. There was no significant effect of either time or diet on the saturated and polyunsaturated n-6 unesterified plasma fatty acids (Fig. 2a and 2b). Monounsaturated unesterified fatty acids did change significantly with time, but did not differ between diet groups [ANOVA, time effect, F(2, 31) ⫽ 6.59, p ⫽ 0.004]. Monounsaturated unesterified fatty acids were highest prior to hibernation (prehibernation versus capture p ⫽ 0.001; prehibernation versus posthibernation p ⫽ 0.021]. Overall, the linseed oil diet group had significantly more unesterified n-3 polyunsaturated fatty acids in the plasma than the control group (ANOVA, diet effect, p ⫽ 0.05), but the concentration of n-3 PUFAs did not change significantly over time. During the summer (Apr–Sept), both diet groups exhibited normal patterns of food intake and body mass changes (Figs. 3 and 4). There were no significant differences in food intake between the two groups during the summer. Food intake increased each week in the control and linseed oil fed groups until it peaked in June, after which time all marmots began to eat less even though ad lib food was still provided. Similarly, the body mass of the marmots on both
Fig. 3. Mean food intake per day each week (⫾1 SE) for marmots on a control and linseed oil diet throughout the experiment. Food intake did not differ between the two groups during the summer. **Indicates a significant difference in food intake between diet groups during the winter. Marmots were housed at 20⬚C on a long-day light cycle (14L:10D) except during winter when they were held in a cold room at 5⬚C/darkness (indicated by arrows).
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Fig. 4. Average monthly body mass (⫾1 SE) of marmots on a control or linseed oil diet. The change in body mass over time was significantly different between the two diet groups. Although both groups gained body mass similarly during the summer, during the winter the control group lost body mass more quickly than the linseed oil diet group.
diets increased until August, at which point body mass began to decline. In October, the marmots were placed under winter conditions in a cold room at 5⬚C in total darkness. As expected, the marmots in the control group ceased feeding, although food was still available. In the linseed oil diet group, five of eight marmots continued to eat for the entire winter (Oct.– March) (Fig. 3). Thus, the food intake differed significantly between the two groups during the winter [repeated-measures ANOVA, diet effect, F(1,11) ⫽ 6.34, p ⫽ 0.029]. Both groups resumed eating in March. This difference in food intake is reflected in the body mass changes during the winter (Fig. 4). Although both groups lost body mass during the winter, the average body mass in the control group declined more rapidly than the body mass of the linseed oil diet group [repeated-measures ANOVA, diet ⫻ time interaction, F(1,9) ⫽ 2.55, p ⫽ 0.044]. Furthermore, four of the marmots fed the linseed oil diet gained body mass from December to January. The time spent in hibernation differed between the two groups. Shortly after being placed in the cold room, all of the control marmots entered their first torpor bout (Tb ⬍ 10⬚C) and continued to hibernate until March. However, only two of the marmots on the linseed oil diet hibernated in the same manner as the control group. Five of the remaining marmots never entered torpor, and one marmot aroused from torpor for the last time in January and began to eat (significant difference in hibernation, Fisher’s Exact Test, p ⫽ 0.021). Figure 5 contrasts the changes in Tb and body mass for a control and a linseed oil diet marmot during the winter. Due to limited plasma, analysis of testosterone and estradiol levels were done on a subset of samples from the linseed oil diet group (n ⫽ 11). Our results did not indicate that marmots on the linseed oil diet had levels of either steroid
Fig. 5. Body temperature and body mass of a representative control marmot (A) and a linseed oil diet marmot (B) under winter conditions (5⬚C/ darkness). Breaks in the line indicate missing data, and spikes in the traces represent radio interferences and do not represent actual changes in Tb.
hormone outside the normal range (testosterone 0.96–2.1 ng/mL; estradiol 0.82–2.55 ng/mL). 4. Discussion This is the first study to have elevated the percentage of ␣-18:3 in the marmot’s WAT and plasma unesterified fatty acids through dietary manipulation prior to hibernation. The WAT of the linseed oil diet marmots was 10% ␣-18:3 (Fig. 1), whereas the control marmots only had a trace (⬍1%) percentage of ␣-18:3 in their WAT. In addition, the linseed oil diet marmots had significantly less 18:2 in their WAT than the control marmots prior to hibernation. Furthermore, the concentration of unesterified ␣-18:3 was significantly higher in the plasma of the linseed oil diet marmots than in the control marmots. Therefore, this study presented the first opportunity to observe how ␣-18:3 is used during hibernation and how an increase in ␣-18:3 in WAT and plasma unesterified fatty acids might alter hibernation behavior. We were unable to observe how ␣-18:3 was used during hibernation because most of the marmots in the linseed oil
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diet did not hibernate and continued to eat during the winter. These results were unexpected for at least two reasons. First, in previous experiments, we have never been able to prevent marmots from hibernating by altering the fatty acid composition of their diet. Second, we have never known marmots to continue eating during the winter, even though food was always available in their cage [3,8,9]. The linseed oil diet was reviewed for possible problems that may have inhibited hibernation. The basic diet formulation, to which linseed oil was added, has been used in our laboratory before, under similar conditions, and no inhibition of hibernation was observed. We can be reasonably sure that linseed oil was not toxic to the marmots as it is regularly used in health and nutritional studies (Purina, personal communication), and often at concentrations two to five times higher than that used in this study, without ill effects [17–24]. In previous hibernation studies there have been three factors implicated in inhibition of hibernation: illness, testosterone, and peroxidation of fatty acids [5,25,26]. None of the marmots in the linseed oil diet group showed signs of illness or infections. Preliminary testosterone and estradiol results indicated that the linseed oil diet group did not have levels of either hormone above normal. Diets rich in ␣-18:3, such as linseed oil, are not considered to significantly increase peroxidation when compared to diets rich in 18:2 [27]; however, teasing apart the effects of ␣-18:3 and lipid peroxidation would require further study. Although plant oils have been routinely used to measure the effects of fatty acids on hibernation [5, 6], we must be cautious in concluding the effects we observed were due strictly to ␣-18:3 and not some other component of linseed oil. This is especially true considering data from field studies on marmots. In midsummer, the WAT of free-ranging marmots can be as much as 25% ␣-18:3, and as already described, their diet contains components rich in ␣-18:3 [9, 10]. Presumably, marmots in the field are able to hibernate. Although this would suggest that ␣-18:3 would not be a likely factor in inhibiting hibernation, a recent field study we conducted [28] indicates that there are many factors that may need to be considered. For example, the concentration of ␣-18:3 in the plants in the marmot’s diet decreased significantly over the summer. Also, at some point either just before or during hibernation, the percentage of ␣-18:3 in the WAT dropped by 50%. Therefore, a reduction in ␣-18:3, either through diet or metabolism, may be necessary for entering into or maintaining hibernation. Marmots in the laboratory always received the same elevated level of ␣-18:3, and consequently, may not have been able to hibernate. The relationship between ␣-18:3, linseed oil and hibernation appears to be complex, and will certainly require further study. Interestingly, there is evidence from other fields that the prostaglandins formed from 18:2 and ␣-18:3 can influence several behaviors including food intake, thermoregulation, and circadian cycles [21, 29–31,35]. It is thought that prostaglandins modify these behaviors via their action
on the hypothalamus [32], which has been implicated in regulation of hibernation behavior as well [33, 34]. Previous research has indicated that 18:2 is retained in fat depots of hibernators and animals with optimal levels of dietary 18:2 are able to conserve more energy during hibernation. Conversely, feeding hibernators a linseed oil diet, with ␣-18:3, resulted in a general inhibition of hibernation and changes in food intake. Further studies will be required to fully separate the effects of linseed oil and ␣-18:3, as well as to understand the separate roles the two essential fatty acids may play in regulating hibernation. Acknowledgments This study was funded by a NSF Grant #IBN9630683 to G.L.F. We would like to thank Dr. Gordon Niswender and the Endocrine Laboratory of Animal Reproduction and Biotechnology for analysis of the testosterone and estradiol samples, and the Metabolic Core Laboratory in the Clinical Nutritional Research Unit at the University of Colorado for the analysis of insulin samples. We would also like to thank Ryan Gill for his assistance on this project. We appreciate the assistance with our statistical analyses from Dr. Jim Zumbrunnen at Colorado State University. References [1] Galli C, Simopooulos AP. Dietary 3 and 6 fatty acids: Biological effects and nutritional essentiality. New York: Plenum Press, 1989. [2] Lehninger AL, Nelson DL, Cox MM. Principles in Biochemistry, 2nd ed. New York: Worth Publishers, 1993. [3] Florant GL, Hester L, Ameenuddin S, Rintoul DA. The effect of a low essential fatty acid diet on hibernation in marmots. Am J Physiol 1993;264:R747–53. [4] Frank CL. The influence of dietary fatty acids on hibernation by golden-mantled ground squirrels (Spermophilus lateralis). Physiol Zool 1992;65:906–20. [5] Frank CL, Storey KB. The optimal depot fat composition for hibernation by golden-mantled ground squirrels (Spermophilus lateralis). J Comp Physiol 1995;164:536–42. [6] Geiser F, Kenagy GJ. Polyunsaturated lipid diet lengthens torpor and reduces body temperature in a hibernator. Am J Physiol 1987;252: R897–901. [7] Geiser F, McAllan BM, Kenagy GJ. The degree of dietary fatty acid unsaturation affects torpor patterns and lipid composition of a hibernator. J Comp Physiol 1994;164:299–305. [8] Thorpe CR, Ram PK, Florant GL. Diet alters metabolic rate in the yellow-bellied marmot (Marmota flaviventris) during hibernation. Physiol Zool 1994;67:1213–29. [9] Florant GL, Nuttle LC, Mullinex DE, Rintoul DA. Plasma and white adipose tissue lipid composition in marmots. Am J Physiol 1990;258: R1123–31. [10] Florant GL, Ameednuddin S, Rintoul DA. Dietary effects on lipid composition and metabolism. In: Rosmos DR, Himms–Hagen J, and Suzuki M, editors. Obesity: Dietary Factors and Control. Tokyo: Japan Soc Sci Press, 1991. pp. 45–57. [11] Frank CL. Adaptations for hibernation in the depot fats of a ground squirrel (Spermophilus beldingi). Can J Zool 1991;69:2707–11. [12] Davis DE. HIbernation and circannual rhythms of food consumption in marmots and ground squirrels. Q Rev Biol 1976;51:477–514. [13] Lepage G, Roy CC. Improved recovery of fatty acid through direct
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transesterification without prior extraction or purification. J Lipid Res 1984;25:1391–6. Christie WW. Gas Chromatography and Lipids. Scotland: The Oily Press, 1989; p. 39. Berndtson WE, Pizkett BW, Nett TM. Reproductive physiology of the stallion IV: seasonal changes in testosterone concentration of peripheral plasma. J Reprod Fertil 1974;39:115–8. England BG, Niswender GD, Midgley AR. Radioimmunoassay of estradiol-17B without chromatography. Clin Endocrinol Metab 1974; 38:42–50. Fritsche KL, Johnston PV. Modulation of eicosanoid production and cell-mediated cytotoxicity by dietary ␣-linolenic acid in BALB/c mice. Lipids 1989;24:305–11. Fritsche KL, Johnston PV. Effect of dietary omega-3 fatty acids on cell-mediated cytotoxicity in BALB/c mice. Nutr Res 1990;10:577–88. Garg ML, Sebovka E, Wierzbicki, A Thomson, ABR, Clandinin MT. Differential effects of dietary linoleic and ␣-linolenic acid on lipid metabolism in rat tissues. Lipids 1988;23:847–52. Jeffery NM, Sanderson P, Sherrington EJ, Newsholme EA, Calder PC. The ratio of n-6 to n-3 polyunsaturated fatty acids in the diet alter serum lipid levels and lymphocyte Marshall LA, Johnston PV. The influence of dietary essential fatty acids on rat immunocompetent cell prostaglandin synthesis and mitogen-induced blastogenesis. J Nutr 1985;115:1572–80. Rustan AC, Christiansen EN, Drevon CA. Serum lipids, hepatic glycerolipid metabolism and peroxisomal fatty acid oxidation in rats fed n-3 and n-6 fatty acids. Biochem J 1992;283:333–9. Takeuchi H, Matsuo T, Tokuyama K, Shimomura Y, Suzuki M. Diet induced thermogenesis is lower in rats fed a lard diet than in those fed a high oleic acid safflower oil diet, a safflower oil diet and a linseed oil diet. J Nutr 1995;125:920–5. Takeuchi H, Matsuo T, Tokuyama K, Suzuki M. Effect of dietary type on -oxidation of brown adipose tissue and Na⫹ channel density of brain nerve membrane in rats. J Nutr Sci Vitaminol 42:161–6.
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[25] Dark J, Miller DR, Zucker I. Gonadectomy in the spring reinstates hibernation in male golden-mantled ground squirrels. Am J Physiol 1996;270:R1240–43. [26] Lee TMK, Pelz P, Licht P, Zucker I. Testosterone influences hibernation in golden-mantled ground squirrels. Am J Physiol 1990;259: R760–7. [27] Kobayashi T, Shimizugawa T, Fukamizu Y, Huang M, Watanabe S, Okuyama H. Assessment of the possible adverse effects of oils enriched with n-3 fatty acids in rats; peroxisomal proliferation, mitochondrial dysfunctions and apoplexy. J Nutr Biochem 1996;7:542–48. [28] Hill VL, Florant GL. Patterns of fatty acid composition in free-ranging yellow-bellied marmots (Marmota flaviventris) and their diet. Can J Zool (in press). [29] Levine AS, Morley JE. The effect of prostaglandins (PGE2 and PGF2␣) on food intake in rats. Pharmacol Biochem Behav 1981;15: 735–8. [30] Marshall LA, Johnston PV. Modulation of tissue prostaglandin synthesizing capacity by increased ratios of dietary ␣-linolenic acid to linoleic acid. Lipids 1982;17:905–12. [31] Wainwright PE. Essential fatty acids and behavior: is there a role for the eicosanoids? In: Yehuda S, and Mostofsky DI, editors. Handbook of Essential Fatty Acids and Biology: Biochemistry, Physiology and Behavioral Neurobiology. Totowa: Humana Press Inc., 1991. pp. 299–341. [32] Baile CA, Simpson CW, Bean SM, Mclaughlin CL, Jacobs HL. Prostaglandins and food intake of rats: a component of energy regulation? Physiol Behav 1973;10:1077–85. [33] Florant GL, Heller HC. CNS regulation of body temperature in euthermic and hibernating marmots (Marmota flaviventris). Am J Physiol 1977;232:R203–8. [34] Wang LCH. Advances in Comparative and Environmental Physiology. Berlin: Springer Verlag, 1989. pp. 361–401. [35] Scales WE, Kluger MJ. Effect of antipyretic drugs on circadian rhythm in body temperature of rats. Am J Physiol 1987;253:R306–13.
Original articles
The effect of a linseed oil diet on hibernation in yellow-bellied marmots (Marmota flaviventris) Vanessa L. Hill, Gregory L. Florant* Colorado State University, Department of Biology, Ft. Collins, CO 80523, USA Received 22 December 1998; received in revised form 5 August 1999; accepted 26 August 1999
Abstract The essential fatty acids (EFAs), ␣-linolenic acid (18:3,n-3) and linoleic acid (18:2,n-6) are known to be important for mammalian hibernation. In marmots (Marmota flaviventris), reducing both dietary EFAs alters hibernation patterns by causing an increase in energy expenditure, but hibernation still occurs. In this study, marmots fed a diet high in ␣-linolenic acid, with normal linoleic acid levels, had significantly (p ⬍ 0.05) more ␣-18:3 in their WAT and plasma unesterified fatty acids after 4 months than did marmots fed a control diet. During the winter, the control marmots hibernated normally while the marmots fed the ␣-18:3 diet did not hibernate, continued to eat, and lost less mass than the control group during the winter. These results suggest that ␣-18:3 may play a role in regulating normal hibernation behavior in marmots. © 2000 Elsevier Science Inc. All rights reserved. Keywords: Hibernation; Essential fatty acids; ␣-Linolenic acid; White adipose tissue; Plasma
1. Introduction The polyunsaturated, essential fatty acid, linoleic acid (cis-9,12 octadecadienoic acid or 18:2,n-6) is important for normal hibernation in mammals that hibernate (i.e., hibernators). Linoleic acid is considered essential because mammals cannot synthesize it, and linoleic acid is essential for normal reproduction, skin maintenance, and other physiologic functions [1,2]. Hibernators that do not obtain sufficient 18:2 in their diet during the summer feeding period store less 18:2 in their white adipose tissue (WAT) and arouse more frequently, have shorter torpor bouts and higher metabolic rates during hibernation than hibernators with sufficient dietary 18:2 [3–8]. These changes in hibernation behavior increase energy expenditure, which might increase the rate at which endogenous energy reserves (i.e., WAT) are depleted, and jeopardize the hibernator’s over winter survival. The WAT of free-ranging hibernators contains another, essential, polyunsaturated fatty acid, ␣-linolenic acid (cis-9,12,15 octadecatrienoic acid or ␣-18:3,n-3) [9–11]. In previous hibernation studies, the diets contained very little ␣-18:3 (⬍3% of total lipids). Because hibernators cannot synthesize ␣-18: 3, the effects of ␣-18:3 in the diet and WAT of hibernators has not been investigated in laboratory experiments [3, 4, 6– * Corresponding author. Tel.: 970-491-7627; Fax: 970-491-0649 E-mail address: [email protected]
8]. Consequently, very little is known about how ␣-18:3, and the longer chain, polyunsaturated fatty acids (n-3, PUFAs) synthesized from ␣-18:3, are stored and utilized for hibernation. Furthermore, it is not known if changes in dietary ␣-18:3, like 18:2, will result in changes in behavior (i.e., torpor bout patterns) during hibernation. In this study, we fed yellow-bellied marmots (Marmota flaviventris) a linseed oil diet with a concentration of ␣-18:3 similar to the leafy portions of plants that are the main components of their natural diet [9]. We compared this group to a group of marmots fed a control diet. Marmots are particularly good animal models to study the effect of dietary fatty acids on hibernation because they do not eat during the winter and rely entirely on fat stored from summer feeding [3, 12]. The first objective of this study is to determine the seasonal patterns of fatty acid composition of WAT and plasma unesterified fatty acids in hibernators fed a diet rich in ␣-18: 3. The WAT composition represents the storage of fatty acids and the plasma unesterified fatty acids are a measure of fatty acid utilization. We hypothesize that ␣-18:3 will be stored in WAT during the summer and preferentially retained in WAT during hibernation as has been shown to happen with the other essential PUFA, 18:2 [3,9]. The second objective of this study is to determine what effect a diet rich in ␣-18:3 has on hibernation behavior as measured by torpor bout length and body temperatures (Tbs). Because we know that the essential PUFA 18:2 in the diet of hibernators results in longer torpor bouts and lower Tbs during hiberna-
0031-9384/00/$ – see front matter © 2000 Elsevier Science Inc. All rights reserved. PII: S0031-9384(99)00 1 7 7 - 8
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tion, we expect that ␣-18:3, which is also an essential PUFA, will have a similar effect. Specifically, we hypothesize that marmots fed the linseed oil diet will have longer torpor bouts and/or lower Tbs than marmots fed a control diet.
2. Materials and methods 2.1. Animals Nine adult marmots of both genders were trapped in Colorado during May and June of 1996, and immediately brought into the lab. These marmots were randomly placed in either the control or linseed oil diet group. From a group of four marmots that had been in the lab for 1 year, two were randomly placed in each group. The control diet group consisted of five marmots fed Purina 5001 and the other group consisted of eight marmots fed a 4.5% linseed oil (Purina 5806-I). Body mass was measured monthly throughout the study. The linseed oil diet was designed to have levels of ␣-18: 3 acid that were higher than the control diet, but comparable to the marmots’ natural diet. The diets were nutritionally equivalent except for the composition of the fat (see Table 1). Both diets were made of all natural ingredients, and meet the requirements established by Purina Research and the National Academy of Sciences (Purina, personal communication). The form of linseed oil used in our diet is standard for many experimental diets, and is safe and nontoxic. The antioxidant, ethoxyquin, was added to the diet to prevent oxidation of fatty acids. The linseed oil diet was also kept frozen or under refrigeration until fed to the marmots to further retard the oxidation of fatty acids. 2.2. Summer conditions Animals were caged individually at 25⬚C under a longday light cycle (14L:10D) during the summer (April–September). Both food and water were provided ad lib. Food intake was measured on a weekly basis. Each day the amount of food fed to each marmot was weighed. Twice per week the food left in the cage (including spillage) was weighed. The amount of food fed each marmot was summed for the week and the amount left in the cage for that week was subtracted. This total was then divided by 7 to calculate the average daily intake.
2.3. Winter conditions In October, the marmots were moved to a 5 ⫾ 3⬚C cold room and complete darkness. Cages for control and linseed oil diet marmots alternated in the cold room. Average daily food intake was calculated every 2 weeks to minimize disturbance to hibernating animals. Marmots were fed every 3 to 4 days, and leftover food was removed once every 2 weeks. 2.4. White adipose tissue and plasma sampling The WAT of each marmot was sampled three times: at capture (Apr–June), prior to hibernation (October), and immediately after hibernation (March). Tissue samples were taken by making a small 1⬙ incision off midline into the abdominal cavity after the marmot had been anesthetized with ketamine (32 mg/kg) mixed with acepromazine (10 mg/kg). A small sample of WAT (⬍1 g) from either the gonadal or omental fat pad was biopsied and immediately placed under nitrogen and stored in a freezer at ⫺80⬚C. The triacylglycerol content and composition of gonadal and omental fat deposits are very similar [9]. Therefore, samples from either depot were used. A blood sample was taken from the femoral artery or vein at the same time that WAT was sampled. The blood was centrifuged, and the recovered plasma stored in a nonheparinized tube in a freezer at ⫺80⬚C. Marmots were allowed 1 week to recover after surgery at 25⬚C (14L: 10D). All procedures were approved by the Colorado State University Animal Care and Use Committee. 2.5. Body temperature/torpor bout measurements In October, temperature sensitive transmitters encased in paraffin (Minimitter, Model T-M Disc with a 2000MAH lithium battery) were surgically implanted into the abdominal cavity of each marmot when WAT and plasma samples were taken. For each animal, the transmitter emitted a specific frequency that was carried via an individual antenna to a computer outside the cold room where the frequencies were converted to body temperatures (Tb) and stored. Body temperatures were recorded for each animal every 30 min from November to March 1996. 2.6. Analysis of WAT and plasma samples Fatty acids were extracted and methylated from a 10–20mg sample of WAT [13]. Separation of fatty acids was
Table 1 Comparison of fatty acid composition of the control and linseed oil experimental diets and leaves from Taraxacum officinale, which is a main component of the marmots’ natural diet Fatty acid (% of total) Diet Control Linseed oil T. officinale
14:0
16:0
16:1
18:0
18:1
18:2
␣-18:3
Other
% Fat
2.1 0.3 0.4
21.8 7.6 14.8
3.8 0.04 2.3
9.5 3.7 1.4
33.4 19.7 2.2
26.1 17.5 17.9
2.1 50.9 58.9
1.2 0.2 1.9
4.5 4.5 3.6
Means are the results of three trials per diet. Percent of each dietary component for control/linseed oil diet: protein 23.4/23.5, fat 4.5/4.5, fiber 5.3/5.3, carbohydrate 66.5/66.4; kcal/g 3.3/3.7 (Purina Test Diets, Inc.).
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achieved with a packed column (10% SP2330 100/120 chromosorb WAW, Supelco, Inc.) on a Hewlett-Packard 5890 gas chromatograph. The temperature of the column was 100⬚C at injection, held there for 1 min, and then the temperature was increased by 5⬚C every minute until it reached 260⬚C, after which time all fatty acids had been eluted. All fatty acids were identified using standards from Matreya, Inc. (Pleasant Gap, PA). Plasma unesterified fatty acids were extracted and methylated as previously described [9], with one modification: unesterified fatty acids were separated from other plasma lipids using a solvent mixture of 80 hexane:20 ethyl ether:2 formic acid [14]. The unesterified fatty acid methyl esters were identified by GC in the same manner as the WAT fatty acids. Unesterified fatty acid concentrations were determined by 50 nmol pentadecanoic acid (15:0) added as an internal standard to each sample.
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ids (PUFA, n-6) — 18:2, 20:3, 20:4, 22:4; polyunsaturated n-3 fatty acids (PUFA n-3) — ␣-18:3, 20:5, 22:5, 22:6. A two-way ANOVA, with diet and time as independent variables, was applied to each class of fatty acids (i.e., SFAs, MUFAs, etc.) in WAT and plasma unesterified fatty acids. If the overall ANOVA model was found to be significant for a particular class of fatty acids, significant differences between time and diets were detected using pairwise comparisons of least squares means. Five preplanned comparisons were used: capture (control diet versus linseed oil diet), capture versus prehibernation (control and linseed oil diet), and prehibernation versus posthibernation (control and linseed oil diet). The significance level of these comparisons was modified with a Bonferroni adjustment (p ⫽ 0.05/5 ⫽ 0.01).
2.7. Identification of fatty acids The following fatty acids were identified in varying amounts in WAT, plasma unesterified fatty acids and the diets: the saturated fatty acids (SFAs) — tetradecanoic acid (14:0), hexadecanoic acid (16:0), octadecenoic acid (18:0), and eicosanoic acid (20:0); the monounsaturated fatty acids (MUFAs) — 9-hexadecanoic acid (n-7,16:1) and 9-octadecenoic acid (n-9,18:1); the polyunsaturated n-6 fatty acids (n-6 PUFAs) — 9,12-octadecadienoic acid (18:2), 8,11,14eicosatrienoic acid (20:3), 5,8,11,14,17-eicosatetraenoic acid (20:4), and 7,10,13,16-docosatetraenoic acid (22:4); the polyunsaturated n-3 fatty acids (n-3 PUFAs) — 9,12,15octadecatrienoic acid (␣-18:3), 5,8,11,14,17-eicosapentaeoic acid (20:5), 7,10,13,16,19-docosapentaenoic acid (22: 5), and 4,7,10,13,16,19-docosahexanoic acid (22:6). 2.8. Hormone analysis Testosterone levels and estradiol levels were analyzed by Dr. Gordon Niswender and the Endocrine Laboratory of Animal Reproduction and Biotechnology [15,16]. 2.9. Statistics All statistical analyses were done with the SAS program (SAS Institute). All data were checked to be sure that the assumptions of the models were not violated. Food intake and mass data between the two diet groups were compared using a repeated measures analysis of variance design. The first 2 months of body mass data were not used in the analysis because the sample sizes for each group were changing as newly trapped animals were added. For this reason, and the fact that the animals needed an additional acclimation period to the lab, the first 3 months of food intake data were not used. For statistical analysis, fatty acids from WAT and plasma were combined into four categories: saturated fatty acids (SFAs) — 14:0,16:0,18:0,20:0; monounsaturated fatty acids (MUFAs) — 16:1, 18:1; polyunsaturated n-6 fatty ac-
Fig. 1. Fatty acid composition of the WAT of control (A) and linseed oil (B) fed marmots at capture (April–June), prior to hibernation (Oct.) and after hibernation (March). All points represent the mean ⫾ 1 SE. Saturated fatty acids (SFAs) included primarily 16:0 with ⬍5% of 14:0 and 18:0 combined. Monounsaturated fatty acids (MUFAs) were largely 18:1(n-9), with ⬍5% 16:1 (n-7). n-6 Polyunsaturated fatty acids (n-6, PUFAs) were entirely 18:2, and n-3 PUFAs were entirely ␣-18:3 (n-3). *Indicates a significant difference from the previous time period. Control marmots n ⫽ 5 and linseed oil diet marmots n ⫽ 8.
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3. Results The WAT fatty acid composition of the control diet group (Fig. 1a) did not differ significantly from the linseed oil diet group (Fig. 1b) at capture. Between capture and prehibernation, several changes in the fatty acid composition of WAT occurred in both diet groups. The saturate percentage of total WAT fatty acids decreased significantly in the linseed oil diet group [ANOVA, diet ⫻ time, F(2,33) ⫽ 13.11, p ⫽ 0.0001; capture versus prehibernation p ⫽ 0.0001], but did not change in the control group. The monounsaturate percentage of total WAT fatty acids increased significantly in the linseed oil diet group [ANOVA, time, F(2,33) ⫽ 14.17, p ⫽ 0.0001; capture versus prehibernation p ⫽ 0.0003, but did not change in the control group. Linoleic acid (18:2,n-6) was ⬎99% of all polyunsaturated n-6 fatty acids (n-6, PUFA) in the WAT of both diet groups. The percentage of total WAT fatty acids comprised of 18:2 decreased significantly in the linseed oil diet group [ANOVA, diet ⫻ time, F(2,33) ⫽ 12.42, p ⫽ 0.0001; capture versus prehibernation p ⫽ 0.0001]), but remained the same in the
Fig. 2. Changes in classes of unesterified fatty acids in plasma of control (a) and linseed oil diet (b) marmots. All points represent the mean ⫾ 1 SE. *Indicates a significant difference from the previous time period. Saturated fatty acids (SFAs) included 14:0, 16:0, 18:0, and 20:0. Monounsaturates (MUFAs) were the same as described in Fig. 4. n-6 Polyunsaturated fatty acids (n-6 PUFAs) were primarily 18:2, with ⬍50 mol/L of 20:3, 20:4, and 22:4 combined. n-3 PUFA fatty acids were primarily ␣-18:3 with ⬍10 mol/L of 20:5, 22:5, and 22:6 combined. Sample sizes are the same as listed in Fig. 4.
control group. Alpha-linolenic acid (␣-18:3,n-3) was ⬎99% of all polyunsaturated n-3 fatty acids (n-3, PUFA) in the WAT of both diet groups. The percentage of total WAT fatty acids comprised of ␣-18:3 decreased significantly in the control group [ANOVA, diet, F(2,33) ⫽ 12.82, p ⫽ 0.001, time, F(2,33) ⫽ 12.88, p ⫽ 0.0001; capture versus prehibernation p ⫽ 0.005], but did not change significantly in the linseed oil diet group. No significant changes in the fatty acid composition of WAT occurred between prehibernation (Oct.) and posthibernation (March) in either diet group. There was no significant effect of either time or diet on the saturated and polyunsaturated n-6 unesterified plasma fatty acids (Fig. 2a and 2b). Monounsaturated unesterified fatty acids did change significantly with time, but did not differ between diet groups [ANOVA, time effect, F(2, 31) ⫽ 6.59, p ⫽ 0.004]. Monounsaturated unesterified fatty acids were highest prior to hibernation (prehibernation versus capture p ⫽ 0.001; prehibernation versus posthibernation p ⫽ 0.021]. Overall, the linseed oil diet group had significantly more unesterified n-3 polyunsaturated fatty acids in the plasma than the control group (ANOVA, diet effect, p ⫽ 0.05), but the concentration of n-3 PUFAs did not change significantly over time. During the summer (Apr–Sept), both diet groups exhibited normal patterns of food intake and body mass changes (Figs. 3 and 4). There were no significant differences in food intake between the two groups during the summer. Food intake increased each week in the control and linseed oil fed groups until it peaked in June, after which time all marmots began to eat less even though ad lib food was still provided. Similarly, the body mass of the marmots on both
Fig. 3. Mean food intake per day each week (⫾1 SE) for marmots on a control and linseed oil diet throughout the experiment. Food intake did not differ between the two groups during the summer. **Indicates a significant difference in food intake between diet groups during the winter. Marmots were housed at 20⬚C on a long-day light cycle (14L:10D) except during winter when they were held in a cold room at 5⬚C/darkness (indicated by arrows).
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Fig. 4. Average monthly body mass (⫾1 SE) of marmots on a control or linseed oil diet. The change in body mass over time was significantly different between the two diet groups. Although both groups gained body mass similarly during the summer, during the winter the control group lost body mass more quickly than the linseed oil diet group.
diets increased until August, at which point body mass began to decline. In October, the marmots were placed under winter conditions in a cold room at 5⬚C in total darkness. As expected, the marmots in the control group ceased feeding, although food was still available. In the linseed oil diet group, five of eight marmots continued to eat for the entire winter (Oct.– March) (Fig. 3). Thus, the food intake differed significantly between the two groups during the winter [repeated-measures ANOVA, diet effect, F(1,11) ⫽ 6.34, p ⫽ 0.029]. Both groups resumed eating in March. This difference in food intake is reflected in the body mass changes during the winter (Fig. 4). Although both groups lost body mass during the winter, the average body mass in the control group declined more rapidly than the body mass of the linseed oil diet group [repeated-measures ANOVA, diet ⫻ time interaction, F(1,9) ⫽ 2.55, p ⫽ 0.044]. Furthermore, four of the marmots fed the linseed oil diet gained body mass from December to January. The time spent in hibernation differed between the two groups. Shortly after being placed in the cold room, all of the control marmots entered their first torpor bout (Tb ⬍ 10⬚C) and continued to hibernate until March. However, only two of the marmots on the linseed oil diet hibernated in the same manner as the control group. Five of the remaining marmots never entered torpor, and one marmot aroused from torpor for the last time in January and began to eat (significant difference in hibernation, Fisher’s Exact Test, p ⫽ 0.021). Figure 5 contrasts the changes in Tb and body mass for a control and a linseed oil diet marmot during the winter. Due to limited plasma, analysis of testosterone and estradiol levels were done on a subset of samples from the linseed oil diet group (n ⫽ 11). Our results did not indicate that marmots on the linseed oil diet had levels of either steroid
Fig. 5. Body temperature and body mass of a representative control marmot (A) and a linseed oil diet marmot (B) under winter conditions (5⬚C/ darkness). Breaks in the line indicate missing data, and spikes in the traces represent radio interferences and do not represent actual changes in Tb.
hormone outside the normal range (testosterone 0.96–2.1 ng/mL; estradiol 0.82–2.55 ng/mL). 4. Discussion This is the first study to have elevated the percentage of ␣-18:3 in the marmot’s WAT and plasma unesterified fatty acids through dietary manipulation prior to hibernation. The WAT of the linseed oil diet marmots was 10% ␣-18:3 (Fig. 1), whereas the control marmots only had a trace (⬍1%) percentage of ␣-18:3 in their WAT. In addition, the linseed oil diet marmots had significantly less 18:2 in their WAT than the control marmots prior to hibernation. Furthermore, the concentration of unesterified ␣-18:3 was significantly higher in the plasma of the linseed oil diet marmots than in the control marmots. Therefore, this study presented the first opportunity to observe how ␣-18:3 is used during hibernation and how an increase in ␣-18:3 in WAT and plasma unesterified fatty acids might alter hibernation behavior. We were unable to observe how ␣-18:3 was used during hibernation because most of the marmots in the linseed oil
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diet did not hibernate and continued to eat during the winter. These results were unexpected for at least two reasons. First, in previous experiments, we have never been able to prevent marmots from hibernating by altering the fatty acid composition of their diet. Second, we have never known marmots to continue eating during the winter, even though food was always available in their cage [3,8,9]. The linseed oil diet was reviewed for possible problems that may have inhibited hibernation. The basic diet formulation, to which linseed oil was added, has been used in our laboratory before, under similar conditions, and no inhibition of hibernation was observed. We can be reasonably sure that linseed oil was not toxic to the marmots as it is regularly used in health and nutritional studies (Purina, personal communication), and often at concentrations two to five times higher than that used in this study, without ill effects [17–24]. In previous hibernation studies there have been three factors implicated in inhibition of hibernation: illness, testosterone, and peroxidation of fatty acids [5,25,26]. None of the marmots in the linseed oil diet group showed signs of illness or infections. Preliminary testosterone and estradiol results indicated that the linseed oil diet group did not have levels of either hormone above normal. Diets rich in ␣-18:3, such as linseed oil, are not considered to significantly increase peroxidation when compared to diets rich in 18:2 [27]; however, teasing apart the effects of ␣-18:3 and lipid peroxidation would require further study. Although plant oils have been routinely used to measure the effects of fatty acids on hibernation [5, 6], we must be cautious in concluding the effects we observed were due strictly to ␣-18:3 and not some other component of linseed oil. This is especially true considering data from field studies on marmots. In midsummer, the WAT of free-ranging marmots can be as much as 25% ␣-18:3, and as already described, their diet contains components rich in ␣-18:3 [9, 10]. Presumably, marmots in the field are able to hibernate. Although this would suggest that ␣-18:3 would not be a likely factor in inhibiting hibernation, a recent field study we conducted [28] indicates that there are many factors that may need to be considered. For example, the concentration of ␣-18:3 in the plants in the marmot’s diet decreased significantly over the summer. Also, at some point either just before or during hibernation, the percentage of ␣-18:3 in the WAT dropped by 50%. Therefore, a reduction in ␣-18:3, either through diet or metabolism, may be necessary for entering into or maintaining hibernation. Marmots in the laboratory always received the same elevated level of ␣-18:3, and consequently, may not have been able to hibernate. The relationship between ␣-18:3, linseed oil and hibernation appears to be complex, and will certainly require further study. Interestingly, there is evidence from other fields that the prostaglandins formed from 18:2 and ␣-18:3 can influence several behaviors including food intake, thermoregulation, and circadian cycles [21, 29–31,35]. It is thought that prostaglandins modify these behaviors via their action
on the hypothalamus [32], which has been implicated in regulation of hibernation behavior as well [33, 34]. Previous research has indicated that 18:2 is retained in fat depots of hibernators and animals with optimal levels of dietary 18:2 are able to conserve more energy during hibernation. Conversely, feeding hibernators a linseed oil diet, with ␣-18:3, resulted in a general inhibition of hibernation and changes in food intake. Further studies will be required to fully separate the effects of linseed oil and ␣-18:3, as well as to understand the separate roles the two essential fatty acids may play in regulating hibernation. Acknowledgments This study was funded by a NSF Grant #IBN9630683 to G.L.F. We would like to thank Dr. Gordon Niswender and the Endocrine Laboratory of Animal Reproduction and Biotechnology for analysis of the testosterone and estradiol samples, and the Metabolic Core Laboratory in the Clinical Nutritional Research Unit at the University of Colorado for the analysis of insulin samples. We would also like to thank Ryan Gill for his assistance on this project. We appreciate the assistance with our statistical analyses from Dr. Jim Zumbrunnen at Colorado State University. References [1] Galli C, Simopooulos AP. Dietary 3 and 6 fatty acids: Biological effects and nutritional essentiality. New York: Plenum Press, 1989. [2] Lehninger AL, Nelson DL, Cox MM. Principles in Biochemistry, 2nd ed. New York: Worth Publishers, 1993. [3] Florant GL, Hester L, Ameenuddin S, Rintoul DA. The effect of a low essential fatty acid diet on hibernation in marmots. Am J Physiol 1993;264:R747–53. [4] Frank CL. The influence of dietary fatty acids on hibernation by golden-mantled ground squirrels (Spermophilus lateralis). Physiol Zool 1992;65:906–20. [5] Frank CL, Storey KB. The optimal depot fat composition for hibernation by golden-mantled ground squirrels (Spermophilus lateralis). J Comp Physiol 1995;164:536–42. [6] Geiser F, Kenagy GJ. Polyunsaturated lipid diet lengthens torpor and reduces body temperature in a hibernator. Am J Physiol 1987;252: R897–901. [7] Geiser F, McAllan BM, Kenagy GJ. The degree of dietary fatty acid unsaturation affects torpor patterns and lipid composition of a hibernator. J Comp Physiol 1994;164:299–305. [8] Thorpe CR, Ram PK, Florant GL. Diet alters metabolic rate in the yellow-bellied marmot (Marmota flaviventris) during hibernation. Physiol Zool 1994;67:1213–29. [9] Florant GL, Nuttle LC, Mullinex DE, Rintoul DA. Plasma and white adipose tissue lipid composition in marmots. Am J Physiol 1990;258: R1123–31. [10] Florant GL, Ameednuddin S, Rintoul DA. Dietary effects on lipid composition and metabolism. In: Rosmos DR, Himms–Hagen J, and Suzuki M, editors. Obesity: Dietary Factors and Control. Tokyo: Japan Soc Sci Press, 1991. pp. 45–57. [11] Frank CL. Adaptations for hibernation in the depot fats of a ground squirrel (Spermophilus beldingi). Can J Zool 1991;69:2707–11. [12] Davis DE. HIbernation and circannual rhythms of food consumption in marmots and ground squirrels. Q Rev Biol 1976;51:477–514. [13] Lepage G, Roy CC. Improved recovery of fatty acid through direct
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